Anti-leishmanial compound and anti-leishmanial drug

ABSTRACT

Provided is an anti-leishmanial compound represented by formula (3):

TECHNICAL FIELD

The present invention relates to an anti-leishmanial compound having ahigh anti-leishmanial activity, and an anti-leishmanial drug.

BACKGROUND ART

Leishmaniasis is an infectious disease caused by becoming infected withthe genus Leishmania. Leishmaniasis has been designated as one of sixmajor tropical diseases by the World Health Organization (WHO).Leishmaniasis is vector-borne disease by the bite of blood-suckingsandfly vectors, resulting in the parasite inoculation to mammalianhosts. The symptoms of Leishmaniasis is fatal in severe ranging frommild to heal, but pentavalent antimony formulations have been usedprimarily as a treatment known to cause severe adverse side effects.Therefore, there is a demand for a new drug having a low risk of adverseside effects. Furthermore, AmBisome has been developed as a drugsuppressing the adverse side effects, but this drug has a problem thatthe drug is expensive.

On the other hand, it has been reported that marine algae-derivedmetabolites exhibit an anti-leishmanial activity (see, for example,Non-Patent Document 1). However, this document does not specify whichcompound derived from marine algae would have an anti-leishmanialactivity.

CITATION LIST

{Non Patent Literature}

{NPL 1} Y. Freile-Pelegrin, D. Robledo, M. J. Chan-Bacab, B. O.Orrtega-Morales, “Antileishmanial properties of tropical marine algaeextract,” Fitoterapia (Holland), Elsevier, 2008, 79, p. 374 to 377

SUMMARY OF INVENTION Technical Problem

The inventors of the present invention conducted a thoroughinvestigation, and as a result, they paid attention to Sargassumyamadae, a brown alga in the Family Sargassaceae of the Order Fucales,and attempted fractionation of an extract of the alga. Accordingly, theinventors found that the extract has a high anti-leishmanial activity,and thus completed the present invention.

Solution to Problem

That is, the anti-leishmanial compound according to the presentinvention is represented by formula (3):

Furthermore, the anti-leishmanial drug according to the presentinvention is characterized by containing the compound represented by theformula (3) and a pharmacologically acceptable salt thereof as activeingredients.

ADVANTAGEOUS EFFECT OF INVENTION

According to the present invention, an anti-leishmanial compound havinga high anti-leishmanial activity and an anti-leishmanial drug can beprovided.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing the procedure of an operation for separationof compounds (first operation) according to the Example of the presentinvention.

FIG. 2 is a diagram showing the procedure of an operation for separationof compounds (first operation) according to the Example of the presentinvention.

FIG. 3 is a diagram showing the procedure of an operation for separationof compounds (first operation) according to the Example of the presentinvention.

FIG. 4 is a diagram showing the procedure of an operation for separationof compounds (first operation) according to the Example of the presentinvention.

FIG. 5 is a diagram showing the procedure of an operation for separationof compounds (first operation) according to the Example of the presentinvention.

FIG. 6 is a diagram showing the procedure of an operation for separationof compounds (second operation) according to the Example of the presentinvention.

FIG. 7 is a diagram showing the procedure of an operation for separationof compounds (second operation) according to the Example of the presentinvention.

FIG. 8 is a diagram showing the procedure of an operation for separationof compounds (second operation) according to the Example of the presentinvention.

FIG. 9 is a graph showing the results of an in vitro anti-leishmanialactivity analysis according to the Example of the present invention.

FIG. 10 is a graph showing the results of an in vivo anti-leishmanialactivity analysis according to the Example of the present invention.

FIG. 11 is a diagram showing the results of an in vivo anti-leishmanialactivity analysis according to the Example of the present invention.

FIG. 12 is a diagram showing the results of an in vivo anti-leishmanialactivity analysis according to the Example of the present invention.

DESCRIPTION OF EMBODIMENTS

Hereinafter, the anti-leishmanial compound and the anti-leishmanial drugaccording to embodiments of the present invention will be described withreference to the attached drawings. The anti-leishmanial compoundaccording to the present invention is at least one of the compoundsrepresented by formulas (1) to (6), and the anti-leishmanial drugaccording to the present invention contains at least one of thecompounds represented by the formulas (1) to (6) as an activeingredient.

In order to recover anyone of the compounds represented by the formulas(1) to (6), those means for separation and purification that aretypically used to collect metabolites may be appropriately utilized onthe algae such as brown algae, while taking into consideration of thephysicochemical properties of the relevant compound. For example, anextraction operation is carried out on the algae, using an organicsolvent, preferably an equivolume solvent mixture ofchloroform-methanol. Subsequently, the compound may be further extractedfrom the extract thus obtained, using an organic solvent such asdichloromethane, or the compound may be adsorbed and eluted usingvarious chromatographic techniques. Furthermore, if necessary, furtherpurification operations may be carried out to recover the compound witha desired purity. In regard to the chromatographic techniques,conventionally used inorganic and organic carriers, for example, silicagel and a polystyrene resin, can be used as the carrier.

The compounds represented by the formulas (1) to (6) are useful as, forexample, antiprotozoal drugs, particularly as anti-leishmanial drugs, inthe field of pharmaceuticals. A compound represented by any one of theformulas (1) to (6) may be used singly, or may be mixed with additivesthat are generally acceptable in formulations and formulated intopreparations. Examples of dosage forms include dosage forms usingperoral preparations such as tablets, granules, capsules, pills,powders, liquids, suspensions, emulsions, syrups, elixirs, and extracts;and dosage forms using parenteral preparations such as injectablepreparations, liquids, suppositories, ointments, patches, poultices, andlotions. However, there are no particular limitations on the dosageform, and the dosage form can be appropriately selected in accordancewith the purpose of therapy, or the like.

In the case of tablets, granules, pills, capsules and powders, additivessuch as an excipient, a binder, a disintegrant, a lubricating agent canbe incorporated therein. Examples of the excipient include starch,carboxymethyl cellulose, sucrose, dextrin, and corn starch.

Examples of the binder include crystalline cellulose, crystallinecellulose carmellose sodium, methylcellulose, hydroxypropyl cellulose,low-substituted hydroxypropyl cellulose, hydroxypropylmethyl cellulose,hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl celluloseacetate succinate, carmellose sodium, ethyl cellulose,carboxymethylethyl cellulose, hydroxyethyl cellulose, wheat starch, ricestarch, corn starch, potato starch, dextrin, pregelatinized starch,partially pregelatinized starch, hydroxypropyl starch, pullulan,polyvinylpyrrolidone, aminoalkyl methacrylate copolymer E, aminoalkylmethacrylate copolymer RS, methacrylic acid copolymer L, methacrylicacid copolymer, polyvinylacetal diethylaminoacetate, polyvinyl alcohol,gum arabic, powdered gum arabic, agar, gelatin, white shellac,tragacanth, purified sucrose, and macrogol.

Examples of the disintegrant include crystalline cellulose, methylcellulose, low-substituted hydroxypropyl cellulose, carmellose,carmellose calcium, carmellose sodium, croscarmellose sodium, wheatstarch, rice starch, corn starch, potato starch, partiallypregelatinized starch, hydroxypropyl starch, carboxymethyl starchsodium, and tragacanth.

Examples of the lubricating agent include wheat starch, rice starch,corn starch, stearic acid, calcium stearate, magnesium stearate,hydrated silicon dioxide, light silicic anhydride, synthetic aluminumsilicate, dry aluminum hydroxide gel, talc, magnesium metasilicatealuminate, calcium hydrogen phosphate, anhydrous calcium hydrogenphosphate, sucrose fatty acid esters, waxes, hydrogenated vegetableoils, and polyethylene glycol.

Furthermore, in the case of liquids, syrups, suspensions, emulsions, andelixirs, a colorant, a taste-masking agent, a flavoring agent and thelike may be incorporated as additives, in addition to inert diluentsthat are generally used, such as water and vegetable oils.

In the case of injectable preparations, additives such as a suspensionliquid, an emulsion liquid, and a ready-to-use solubilizing agent can beincorporated. Furthermore, in the case of ointments and suppositories,fats, fatty oil, lanolin, petrolatum, paraffin, waxes, resins, plastics,a base, a glycol, a higher alcohol, water, an emulsifier, a suspendingagent, and the like can be incorporated as additives. In the case ofpoultices, glycerin, water, a water-soluble polymer, a water-absorptivepolymer, and the like can be incorporated as additives. In the case oflotions, a solvent, an emulsifier, a suspending agent, and the like canbe incorporated as additives.

The anti-leishmanial compound of the present invention can be added to afood product, a chewing gum, a beverage or the like, and then beincorporated into so-called foods for specified health uses (forexample, anti-leishmanial foods), dietary supplements, and the like.

As well, the compounds represented by the formulas (1) to (6) asdescribed above are conceptually considered to include pharmacologicallyacceptable salts of these compounds. That is, the present inventionincludes biochemical precursors that are converted to the compounds andamides by metabolism in the body of a human being or an animal, andexhibit a pharmacological activity. According to the present invention,the term pharmacologically acceptable salt is a salt that is obtained bytreating one of the compounds described above with an acid or a base,and means a salt which does not have significant toxicity and can beused as a medicine. Examples of such an acid addition salt includeaddition salts based on inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, and phosphoric acid; and organic acidssuch as maleic acid, fumaric acid, tartaric acid, and citric acid.Examples of a salt based on a base include salts based on alkali metalhydroxides such as sodium hydroxide and potassium hydroxide; alkalineearth metal hydroxides such as calcium hydroxide and magnesiumhydroxide; and organic bases such as guanidine, triethylamine, anddicyclohexylamine.

EXAMPLES

Hereinafter, the present invention will be more specifically describedby way of Examples.

-   1. Operation for Separation of Compounds Represented By Formulas (1)    to (6)    (1) Extraction of Lipid Components

Sargassum yamadae (294.4 g), a brown alga in the Family Sargassaceae ofthe Order Fucales, was immersed for one day in an equivolume solventmixture (1.4 L) of methanol and chloroform. The residue was removed bysuction filtering, and lipid components were extracted. This residue wasfurther immersed for one day in an equivolume solvent mixture (1.4 L) ofmethanol and chloroform, and lipid components were extracted byperforming suction filtration. The solvent was removed under reducedpressure, and the extract (31.2 g) thus obtained was partitioned intotwo layers using water and chloroform. Thus, lipid components (23.8 g)were obtained. In addition, Sargassum was washed with artificialseawater after collection, dried in a constant temperaturedehumidification chamber, pulverized, and then stored at −20° C. untilused in experiments.

(2) First Operation of Separation

Among the lipid components obtained by the operation described insection (1), 5.14 g was fractionated into sixteen fractions, such asfraction A to fraction P, by open column chromatography (Φ7×45 cm) whilethe proportion of the solvent of an ethyl acetate-hexane system wasvaried, as shown in FIG. 1.

Among these, as shown in FIG. 2 to FIG. 5, Fractions E, K, N and O werefurther subjected to open column chromatography, thin layerchromatography, and HPLC using an ethyl acetate-hexane mixed eluate, andthus various kinds of compounds were separated.

As shown in FIG. 2, the fraction E was subjected to thin layerchromatography, and various kinds of fractions were obtained.

Among these, a compound represented by formula (5) was obtained fromfraction f. Further, fraction e was subjected to HPLC, and the compoundrepresented by the formula (5) was obtained at a retention time of 18minutes. As well, in FIGS. 2 to 8, the masses (mg) of the fractions thusobtained are indicated below the fraction names. Furthermore, in thecase of any of the compounds represented by the formulas (1) to (6), thenumeral of the formula of the relevant compound is indicated in a boldletter. For the fractions obtained by performing HPLC, the retentiontime and the mass (mg) of the compound thus obtained are indicated inorder from the top, and in the case of any of the compounds representedby the formulas (1) to (6), the numeral of the formula of the relevantcompound is indicated in a bold letter.

Furthermore, as shown in FIG. 3, the fraction K was subjected to HPLCusing a 20% ethyl acetate-hexane eluate, and sargaquinoic acid wasobtained at a retention time of 28 minutes, while the compoundrepresented by the formula (1) was obtained at a retention time of 31minutes. As shown in FIG. 4, the fraction N was subjected to thin layerchromatography and HPLC using a 20% ethyl acetate-hexane eluate, and thecompound represented by the formula (2) (HREIMS m/z 440.2560 {M+},C₂₇H₃₆O₅ (Δ−0.3 mmu)) was obtained during HPLC at a retention time of 28minutes. Furthermore, as shown in FIG. 5, the fraction O was subjectedto HPLC using a 55% ethyl acetate-hexane eluate, and the compoundrepresented by the formula (3) (HREIMS m/z 440.2934 {M+}, C₂₈H₄₀O₄(Δ+0.8 mmu) was obtained at a retention time of 28 minutes.

(3) Second Operation of Separation

Among the lipid components obtained by the extraction operation of lipidcomponents as described above, the remaining 18.6 g, which was not usedin the first operation of separation, was fractionated into fifteenfractions, such as fraction A′ to fraction O′, by open columnchromatography (Φ7×45 cm) as shown in FIG. 6, while the proportion ofthe solvent of an ethyl acetate-hexane system was varied.

As shown in FIG. 7, among the fractions thus obtained, fraction C′ andfraction D′ were combined, and the mixture was subjected to extractionby means of Sep-Pak. A fraction which had been extracted using a 0.5%ethyl acetate-hexane mixed eluate, was subjected to HPLC using a 1%ethyl acetate-hexane eluate, and the compound represented by the formula(6) was obtained at a retention time of 182 minutes. Furthermore, afraction which had been extracted using a 1% ethyl acetate-hexane mixedeluate, was subjected to HPLC using a 7% ethyl acetate-hexane eluate,and the compound represented by the formula (5) was obtained at aretention time of 108 minutes.

As shown in FIG. 8, the fraction E′ was subjected to thin layerchromatography, and was further subjected to HPLC using a 20% ethylacetate-hexane mixed eluate. The compound represented by the formula (5)was obtained at a retention time of 40 minutes, and the compoundrepresented by the formula (4) (EIMS m/z 408 C₂₇H₃₆O₃ {M+}, m/z 175 {M−C₁₅H₂₅O}) at retention time of 76 minutes.

The compounds thus obtained were subjected to an analysis of themolecular structures by ¹H-NMR, ¹³C-NMR and the like. The NMR spectraldata are presented in the Tables below. As well, the NMR data of thecompound represented by the formula (1) are presented together with theNMR data published in Bull. Chem. Soc. Jpn., 2008, 81(9), 1125 to 1130,and the NMR data of the compound represented by the formula (4) arepresented together with the NMR data of sargachromenol, which has asimilar structure. The NMR data of the compounds represented by theformulas (5) and (6) are presented together with the NMR data ofsargaquinoic acid.

TABLE 1 NMR data of compound represented by formula (1) (1) literature #δ_(H) J (Hz) δ_(C) δ_(H) J (Hz) 1 187.8 2 147.8 3 6.47 d 1.8 132.5 6.47m 4 187.8 5 6.56 m 133.2 6.57 dq 1.8, 1.4 6 146.0 7 2.07 d 1.4 16.0 2.07d 1.4 1′ 3.17 d 7.3 27.8 3.16 br. d 7.3 2′ 5.24 br. d 7.3 121.2 5.24 tq7.3, 1.4 3′ 136.2 4′ 2.94 d 7.3 42.4 2.94 br. d 6.9 5′ 6.75 td 6.9, 16.0145.0 6.75 td  6.9, 16.0 6′ 6.09 br. d 132.5 6.09 dt 16.0, 1.4  7′ 198.38′ 2.27 27.1 2.26 s 9′ 1.67 br. s 16.4 1.67 br. s * not determined

TABLE 2 NMR data of compound represented by formula (2) # δ_(C) δ_(H)J(Hz) HMBC(C)  1 187.7  2 145.8  3 134.6 6.64 br.d 2.7  4 188.4  5 133.36.57 m  6 145.9  7 16.1 2.05 d 1.4 1, 5, 6  1′ 36.0 2.78 dd 14.2, 3.7 2,3, 2′ 2.54 dd 14.2, 7.1  2′ 73.3 4.26 dd 8.7, 3.7 2′  3′ 150.8  4′ 26.42.18 overlapped 5′  5′ 31.7 2.20 overlapped 4′, 20′  6′ 124.3 5.15 dq6.4, 0.9  7′ 135.0  8′ 39.0 2.08 overlapped 6′  9′ 28.3 2.58 q 7.3 10′,11′ 10′ 144.0 5.95 t 7.3 8′, 12′ 11′ 130.5 12′ 34.6 2.27 t 7.8 13′, 14′13′ 27.9 2.14 overlapped 14′ 123.4 5.10 overlapped 15′ 132.3 16′ 25.61.68 s 17′ 17′ 17.7 1.58 s 16′ 18′ 166.0 19′ 16.1 1.62 s 6′, 7′, 8′ 20′110.2 5.08 s 4′ 4.90 s

TABLE 3 NMR data of compound represented by formula (3) (3) # δ_(C)δ_(H) J(Hz) HMBC(C)  1 200.7  2 50.2  3 46.5 2.72 d 16.5 1, 2, 4, 5 3.28d 16.5 1, 2, 4, 5  4 197.4  5 140.8 6.69 d 10.5 4, 6  6 139.9 6.72 d10.5 1, 2, 5  1′ 38.2 2.21 dd 7.8, 13.7 1, 2, 2′, 3′ 2.31 dd 8.2, 13.71, 2, 2′, 3′  2′ 116.7 4.95 dd 7.8, 8.2 4′, 20′  3′ 141.6  4′ 39.7 1.97m 2′, 3′, 5′, 6′  5 26.7 2.04 m 3′, 4′, 6′, 7′  6′ 124.6 5.06 t 6.9 4′ 7′ 135.5  8′ 39.6 1.98 m 6′, 7′, 9′, 19′  9′ 26.6 2.05 m 8′, 10′, 11′10′ 124.1 5.10 t 6.9 8′, 18′ 11′ 135.0 12′ 39.9 1.95 m 10′, 11′, 13′,18′ 13′ 26.3 2.05 m 11′, 12′, 14′, 15′ 14′ 124.4 5.10 t 6.9 12′, 16′,17′ 15′ 131.3 16′ 25.7 1.68 s 14′, 15′, 17′ 17′ 17.6 1.60 s 14′, 15′,16′ 18′ 16.0 1.58 s 10′ 19′ 16.0 1.58 s 7′ 20′ 16.4 1.53 s 3′ 21′ 39.72.33 d 17.9 1, 2, 22′ 3.09 d 17.9 22′ 176.0

TABLE 4 NMR data of compound represented by formula (4) sarga- (4)chromenol # (_(C) δ_(H) J (Hz) HMBC (C) δ_(c) δ_(H)  2 77.7 77.6  3130.6 5.57 d 9.6 2, 4a 130.4 5.57  4 122.9 6.26 d 9.6 2, 4a, 5, 8a 122.96.24  4a 121.3 121.2  5 110.3 6.33 d 2.7 4, 7, 8a 110.3 6.32  6 148.5148.9  7 117.1 6.47 d 2.7 5, 8a, 9 117.1 6.48  8 126.3 126.1  8a 144.9144.5  9 15.5 2.14 s 7, 8, 8a 15.4 2.13 10 25.9 1.37 s 2, 3, 1′ 25.81.36  1′ 40.7 1.68 overlapped 2, 10, 2′ 40.7 1.68  2′ 22.6 2.15overlapped 1′, 3′ 22.5  2.1 *  3′ 125.5 5.16 br. t 7.3 2′, 5′, 16′ 124.95.15  4′ 133.6 134.2  5′ 38.3 2.12 t 7.3 4′, 6′, 7′, 16′ 39.0 2.07  6′27.0 2.43 br. q 7.3 4′, 5′, 7′, 8′ 28.0 2.60  7′ 155.5 6.41 t 7.3 5′,6′, 9′, 15′ 145.3 6.00  8′ 143.2 130.6  9′ 24.2 2.25 br. t 7.8 7′, 8′,10′, 15′ 34.4 2.27 10′ 27.4 2.03 br. q 7.3 8′, 9′, 12′ 27.8 2.13 11′123.6 5.09 tt 7.3, 1.4 13′, 14′ 123.4 5.09 12′ 133.6 132.1 13′ 25.7 1.67s 11′, 12′, 14′ 25.5 1.68 14′ 17.6 1.56 s 11′, 12′, 13′ 17.6 1.59 15′195.2 9.34 s 8′ 173.0 16′ 15.8 1.60 s 3′, 4′, 5′ 15.6 1.58 * notdetermined

TABLE 5 NMR data of compound represented by formula (5) sargaquinoic (5)acid # δ_(C) δ_(H) J (Hz) HMBC (C) δ_(C) δ_(H)  1 187.9 187.9  2 154.9148.4  3 133.1 6.45 dt 2.3, 1.8 1 or 4 132.2 6.46  4 188.0 188.0  5133.9 6.54 br. q 1.4 133.1 6.54  6 148.4 145.9  7 16.0 2.06 d 1.4 1, 5,6 15.9 2.05  1′ — 3.13 d 7.3 1, 2, 3, 2′, 3′ — 3.13  2′ 118.2 5.15 br. t7.3 118.0 5.15  3′ 143.2 139.7  4′ 39.5 2.1 * overlapped 39.5 2.1  5′25.7 2.1 * overlapped 26.3 2.1  6′ 125.1 5.09 br. t 7.3 124.4 5.12  7′139.7 134.5  8′ 38.3 2.1 * overlapped 39.0 2.1  9′ 27.4 2.45 q 7.3 8′,10′, 15′ 28.1 2.60 10′ 145.9 6.41 t 7.3 4′, 8′ 145.1 6.01 11′ 132.3130.7 12′ 27.5 2.26 t 7.3 10′, 15′ 34.5 2.26 13′ 27.0 2.1 * overlapped27.8 2.1 14′ 123.6 5.15 br. t 7.3 123.4 5.09 15′ 132.3 132.1 16′ 25.71.67 s 14′, 17′ 25.6 1.67 17′ 17.6 1.57 s 14′, 16′ 17.6 1.58 18′ 195.19.35 s 172.8 19′ 16.1 1.63 s 8′ 15.8 1.60 20′ 16.0 1.64 s 2′, 3′ 16.01.62 * not determined

TABLE 6 NMR data of compound represented by formula (6) 5 sargaquinoicacid # δ_(H) J (Hz) δ_(C) δ_(H) J (Hz) HMBC (C)  1 187.9  2 148.4  36.46 dt 1.6, 2.8 132.2 6.46 dt 1.6, 2.8 4, 5, 1′  4 188.0  5 6.54 dq2.8, 1.4 133.1 6.54 dq 2.8, 1.4 3, 4, 7  6 145.9  7 2.05 d 1.4 15.9 2.05d 1.4 1, 5, 6  1′ 3.13 br. d 7.3 27.5 3.13 br. d 7.3 1, 2, 3, 2′, 3′  2′5.15 overlapped 118.0 5.15 overlapped 1′, 20′  3′ 139.7  4′ 1.9~2.1 *overlapped 39.5  2.1 * overlapped 2′, 3′, 5′  5′ 1.9~2.1 * overlapped26.3  2.1 * overlapped 4′, 6′  6′  5.1 * overlapped 124.4 5.12overlapped 5′, 8′, 19′  7′ 134.5  8′ 1.9~2.1 * overlapped 39.0  2.1 *overlapped 7′, 9′  9′ 1.9~2.1 * overlapped 28.1 2.60 dt 7.3, 7.3 8′,10′, 11′ 10′  5.1 * overlapped 145.1 6.01 t 7.3 8′, 9′, 12′, 18′ 11′130.7 12′ 1.9~2.1 * overlapped 34.5 2.26 br. t 7.6 10′, 11′, 13′, 14′,18′ 13′ 1.9~2.1 * overlapped 27.8  2.1 * overlapped 12′, 14′ 14′ 5.1 *overlapped 123.4 5.09 overlapped 13′, 16′, 17′ 15′ 132.1 16′ 1.67 br. s25.6 1.67 br. s 14′, 15′, 17′ 17′  1.6 * br. s 17.6 1.58 br. s 14′, 15′,16′ 18′  1.6 * br. s 172.8 19′  1.6 * br. s 15.8 1.60 br. s 6′, 7′, 8′20′ 1.62 br. s 16.0 1.62 br. s 2′, 3′, 4′ * not determined

From these NMR data, it was found that the compounds thus obtained arerespectively represented by the formulas (1) to (6) described above.

-   2. Analysis of Anti-leishmanial Activity    (1) Analysis of In Vitro Anti-leishmanial Activity

The compounds represented by formulas (1) to (6), which had beenisolated by the procedure described above, were subjected to an analysisof the physiological activity under the conditions that are describedbelow. Leishmania major promastigotes were selected as the Leishmaniaprotozoa, and a L. major/egfp promastigotes in which a fluorescentprotein egfp gene had been introduced was used. The compounds mentionedabove were used as samples. Thus, the growth inhibition rate wasdetermined under the following conditions.

(Analysis Procedure)

L. major/egfp promastigotes were maintained at 25° C. using a 96-wellplate, in 199 medium (NISSUI Pharmaceutical) containing 10% fetal bovineserum 25 mM and Hepes buffer (ICN Biomedeivals, Inc., Aurora, Ohio) and10 μg/mL Tunicamycin (SIGMA-ALDRICH, inc., USA). Subsequently, to eachwell of 96-well plates containing 100 μL of L. major/egfp suspensionwith 1×10⁶ cells/mL, 100 μL of test solution (sample dissolved in DMSO)was added, and the plates were incubated at 25° C. for 72 h. Thereafter,Fluorescence signals of L. major/egfp promastigotes were measured with afluorescence microplate reader (Fluoroscan Ascent FL, DainipponPharmaceutical Co.) with excitation at 485 nm and emission at 538 nm.Furthermore, to determine the growth inhibition rate of L. major/egfp itis defined as 100% when Amphotericin B was used as the positive control,and the inhibition rate of various samples are presented.

FIG. 9 shows the growth inhibition rate determined as a result of theanalysis. According to these results, it was found that the compoundsrepresented by the formulas (1) to (6) have a physiological activitycharacteristic of inhibiting the growth of the Leishmania protozoa.Particularly, the growth inhibition rate of the compound represented byformula (3) was almost equal to the growth inhibition rate ofAmphotericin B.

(2) Analysis of In Vivo Anti-leishmanial Activity

In order to evaluate the in vivo anti-leishmanial activity of thecompound represented by formula (3), which had been isolated by theprocedure described above, a leishmaniasis mouse model was used toinvestigate the therapeutic effect.

Six-week old mice (Balb/c, male, 6 animals per group) were infected with1×10⁷ cells of cultured promastigotes of L. major PM2. From the dayafter the infection day, the compound represented by the formula (3) wasadministered to each specimen in an amount of 200 [μg by peritonealinjection. The administration schedule was such that the compound wascontinuously administered once a day for three weeks. Further, ascontrol, the same analysis was carried out also for a treatment group towhich Amphotericin B was administered, and for a treatment group towhich none was administered.

The results obtained by measuring the size of ulcer in the mice arepresented in FIG. 10. Furthermore, FIG. 11 shows the fluctuation ofantibody titer in blood of the mice. FIG. 12 shows the results obtainedby performing electrophoresis of a DNA product which has been amplifiedby PCR carried out using the DNA extracted from the mouse blood as atemplate and using the protozoan gene as a target. That is, FIG. 12shows the results for the detection of the protozoan DNA in blood, andwhen the protozoan DNA is present, a PCR product is observed at theposition of 310 bp. As the white color becomes more intense in FIG. 12,it shows that the protozoan DNA is present at a higher concentration. Onthe other hand, when the position of 310 by is black, it shows that theprotozoan DNA is not present.

From these results, it was found that the compound represented by theformula (3) exhibits an anti-leishmanial activity that is equal to thatof Amphotericin B, even in vivo.

1. An isolated anti-leishmanial compound represented by formula (3):


2. An anti-leishmanial drug comprising the isolated anti-leishmanialcompound according to claim 1 or a pharmacologically acceptable saltthereof as an active ingredient.